Ultrasound contrast is a very useful and widely used medical diagnostic technique. The technique takes advantage of the fact that the various fluids and tissues in the body reflect sound waves differently. This results in a contrast between reflected waves that can be detected and used to form an image of the tissue. Ultrasound is used for many different diagnostic purposes, e.g., prenatal imaging or to image bloodflow in the heart and arteries and observe blockages in blood circulation.
It has been discovered that an ultrasound image can be greatly enhanced by the presence of ultrasound contrast agents. By placing such contrast agents within the tissue to be imaged, a greater difference in the reflectance of the sound waves between the tissue to be imaged and the surrounding tissue occurs. This allows much sharper delineation of tissue boundaries and perfusion to be observed.
Such contrast agents are based on the acoustic impedance mismatch between a gas and a liquid. These agents are typically micron-sized bubbles containing various gases encapsulated in polymers, surfactants, proteins, polyaminoacids and their derivatives, liposomes, or inorganic shells. The bubbles, often called “microbubbles”, are typically smaller than 10 micrometers so that they will pass through small vessels such as the capillary bed of the lung and reach the heart. They are commonly filled with a gas because it has been found that these gas-filled microbubbles provide very efficient ultrasound contrast, much better than that observed using liquid or solid contrast particles of equivalent size.
Although non-encapsulated gas microbubbles may be used for some purposes, they tend to change size very rapidly. The larger non-encapsulated gas microbubbles grow while the smaller microbubbles continue to diminish in size. Those that are small enough to pass through the lungs are effectively to small and dilute to provide useful contrast upon reaching the heart and are thus not practical for imaging the left side of the heart. The rate at which microbubbles undergo this type of change depends upon the actual gas used. Less soluble and diffusive types of gas will form non-encapsulated microbubbles that may be usable for imaging the left side of the heart.
Several encapsulated contrast agents have already been developed and are widely used. U.S. Pat. No. 5,614,169 describes microbubbles comprising carbohydrates and amphiphilic C22-50 organic acids. The organic acids were described as preferably containing at least one carboxyl group and could be, e.g., straight chain fatty acids. U.S. Pat. No. 5,352,436 describes microbubbles which are stabilized by the presence of two different surfactants. The first surfactant tends to be substantially soluble and nonionic, examples being polyoxyethylene fatty acid esters, such as polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan nonostearate, polyoxyethylene sorbitan tristearate, polyoxyethylene sorbitan monooleate and mixtures thereof. These esters include TWEEN 20, TWEEN 40, TWEEN 60, TWEEN 65 and TWEEN 80. The second surfactant is preferably insoluble and nonionic. These include sorbitan fatty acid esters, such as sorbitan monostearate, sorbitan monopalmitate and mixtures thereof. Such mixtures include SPAN 40 and SPAN 60, which comprise palmitic acid, myristic acid and pentadecanoic acid, and stearic acid, palmitic acid and myristic acid, respectively. Published PCT patent application WO 96/39197 describes stabilized ultrasound compositions comprising fluorinated amphiphilic compounds wherein these compounds can comprise short to long chain alkyls or fluoroalkyls.
Other patents have also described the use of sugars in ultrasound contrast agents. These include U.S. Pat. Nos. 4,681,119, 4,442,843 and 4,657,756 which disclose the use of particulate solids having a plurality of gas-filled voids and preferably also a plurality of nuclei for microbubble formation. EP-A-0123235 and EP-A-0122624 suggest ultrasound contrast agents consisting of surfactant-coated or surfactant-containing gas-containing microparticles which may include a variety of sugars. DE-A-3834705 suggests the use of suspensions containing microparticles of mixtures of at least one C10-20 fatty acid with at least one non-surface active substance, including sugars such as cyclodextrins, monosaccharides, disaccharides or trisaccharides, as well as other polyols and inorganic and organic salts.
Microbubbles have also been encapsulated in gelatin and albumen. ALBUNEX® Contrast Agent is a suspension of stable microencapsulated air bubbles which are encapsulated in human serum albumin. OPTISON® Contrast Agent is a suspension of stable microencapsulated octafluoropropane bubbles which are encapsulated in human serum albumin. Both are prepared by sonicating dilute human albumin at a temperature slightly below denaturing. ALBUNEX is prepared by sonicating in the presence of air; OPTISON® in the presence of octafluoropropane. Both are composed of gas-filled microbubbles with a mean diameter in the range of 3-5 microns and stabilized by a thin albumin shell.
U.S. Pat. No. 4,684,479 discloses surfactant mixtures for the production of stable gas-in-liquid emulsions comprising: (a) a glycerol monoester of saturated carboxylic acids containing from about 10 to about 18 carbon atoms or aliphatic alcohols containing from about 10 to about 18 carbon atoms; (b) a sterol-aromatic acid ester; and (c) a sterol, terpene, bile acid or alkali metal salt of a bile acid.
U.S. Pat. No. 4,466,442 discloses a solution for the production of gas microbubbles which contains a solution of at least one tenside and at least one viscosity-raising compound. Examples of suitable non-ionic tensides include polyoxyethylene fatty acid esters, and polyoxyethylated sorbitan fatty acid esters. Examples of viscosity-raising compounds include mono- or polysaccharides, dextrans, cyclodextrins, hydroxyethyl amylose, polyols, proteins, proteinaceous materials, amino acids, and blood surrogates.
U.S. Pat. No. 5,573,751 teaches the advantage of using any of a variety of gases in microbubbles which gases are longer lasting than other gases such as air. U.S. Pat. No. 5,352,436 teaches one process for preparing stabilized gas microbubbles with mean diameter less than 10 micrometers. U.S. Pat. No. 5,656,211 also teaches methods for preparing gas-filled microbubbles.
European Patent Application 0231091 discloses emulsions of oil in water containing highly fluorinated organic compounds as contrast agents. U.S. Pat. No. 4,900,540 describes the use of phospholipid-based liposomes containing a gas or gas precursor as a contrast-enhancing agent.
Ultrasound contrast agents have also been used for purposes other than tissue imaging, e.g., measuring pressure in a system or measuring fluid flow rates. U.S. Pat. No. 4,265,251 teaches a method of determining the pressure within a liquid containing vessel by (1) adding a solid precursor for at least one bubble the liquid, (2) retaining the precursor in the liquid for a sufficient time to form at least one bubble and to generate a sonic signal, (3) measuring a characteristic of the sonic signal which is representative of the pressure in the liquid, and (4) determining the pressure in the liquid from the measured characteristic. U.S. Pat. No. 4,483,345 teaches a method of non-destructively measuring the pressure of a desired region within a substance, from outside, by a process wherein ultrasonic waves are applied to the desired region within a substance, to generate bubbles within the liquid existing in the region during the negative pressure cycle of the ultrasonic waves and, thereafter, the generation of bubbles is detected by harmonic or subharmonic ultrasonic waves which accompany such bubbles and/or by the echo of other ultrasonic waves of higher frequency applied to the region. U.S. Pat. No. 4,316,391 teaches a method of measuring fluid flow rate in a system having a conduit through which a fluid flows. The method comprises adding a substance which provides a plurality of bubbles of a known quantity and size to the system upstream of the conduit. A sonic pulse is generated from a position opposite and spaced from the conduit as the bubbles pass through the conduit. Reflected sonic images are measured, which images are created by reflection of the pulse from the wall of the conduit distal from the position. Additionally, other reflections are measured from the bubbles themselves flowing between the two walls. The fluid flow rate is then determined from the sonic images.
A variety of methods are used to make microbubbles. Several of these are outlined in published PCT application WO 96/39197. This same application also describes many of the gases which may be included within the microbubbles.
These known agents are satisfactory for some applications but have limitations that prevent them from being useful for many purposes. They are often not stable when subjected to sterilization procedures and have relatively short half-lives after formulation or reconstitution. Also, the physical properties of the microbubbles at effective concentrations limit the depth in the tissue where a diagnostic image can be obtained. Often useful images can only be acquired for tissue a few centimeters from the ultrasound transmitter/receiver thus limiting the “penetration” of the procedure and the depth in the body from which images can be obtained. The amount of signal loss accountable to a variety of scattering processes is known as attenuation. Further, the physical properties of the microbubbles cause relatively high attenuation that limits the usefulness of the agent to obtain certain types of images, e.g. myocardial perfusion and images that must be obtained immediately after injection.
Additionally, known agents have not proven useful for measuring blood pressure in the body, particularly for measuring pressure within the cardiovascular system.
To illustrate these limitations, consider how an ultrasound image is obtained. An ultrasound image is obtained when an emitted sound wave strikes a microbubble and is reflected back to and detected by a receiver. Attenuation processes occur along the path transversed by the sound wave as it is returning to the receiver. Attenuation increases with the number and size of the microbubbles to which the transmitted signal interacts. Therefore, at some concentration for a given agent, image resolution and contrast is lost. Thus, the image detail deteriorates since many reflected signals no longer reach the receiver at the frequency of imaging. The depth of penetration by the signal is thus limited.
In addition, known ultrasound contrast agents are often administered to a patient by quickly injecting a large volume of the agent into the patient, i.e. a “bolus” injection. Ultrasound measurements are started immediately and taken over the next several seconds or minutes. However, the image obtained from the ultrasound measurement occurs in three stages. The measurement initially produces an image of relatively low diagnostic value as the microbubbles first enter the system. Next, as the concentration of the microbubbles increases, image contrast is lost due to the effects of attenuation. At this stage the image appears to “darken” to a point where virtually no image can be obtained. This shadowing is principally due to attenuation of the ultrasound signal caused by the high concentration of the contrast agent. The numerous microbubbles scatter or translate the reflected signal and prevent it from reaching the receiver. Then, in the third stage, the faded image returns as attenuation abates with a decline in the concentration of the contrast agent due to dilution, destruction, and uptake processes in the imaged organism. Diagnostic imaging is usually carried out in this often brief third stage.
The problem is that in the second stage regions which contain a large number of microbubbles obscure adjacent farfield locations of key medical interest. These adjacent locales often contain relatively much less contrast agent at peak dose. As the overall concentration declines and the image returns, the amount of contrast remaining in these areas of interest is insufficient or sub-optimal for giving good contrast or flow information. For example, myocardial perfusion tests using known contrast agents give poor results, if at all. The high concentration of contrast agent in the heart chambers following a bolus injection initially attenuates the signal obscuring the image of the perfusion of the heart muscle. As the concentration of contrast agent in the blood declines, the attenuation diminishes but little or no contrast enhancement of the heart muscle remains. Similarly, when measuring heart muscle function in a stress test, the patient is “stressed” and the ultrasound image is taken immediately before the patent's heart rate declines. Known ultrasound contrast agents are of limited use during a stress test because the imaging is carried out when these agents are in the second or “attenuated” stage. By the time the image returns, the heart rate is off peak and the image is no longer diagnostic.
Therefore, there is a need for ultrasound contrast agents that are more stable generally than known agents, that permit greater signal penetration than known agents by lowering attenuation, and that are useful in procedures where known agents have limited applicability. Such agents must be highly efficient. They must be very reflective yet minimally attenuating so that imaging procedures can produce better and more consistent ultrasound images, particularly for imaging heart perfusion, measuring blood pressure, and similar procedures.